Designed multi-domain protein as a carrier of nucleic acids into cells.
Protein-based nucleic acid carriers offer attractive possibilities to enhance in vitro and in vivo gene delivery to combat diseases. A multi-domain fusion protein, namely TAT-NLS-Mu, designated as TNM, has been designed, cloned, heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. The recombinant chimera TNM harbors three epitopes, a cell-penetrating (TAT) domain, a nuclear localization domain comprising of three nuclear localization sequence (NLS) motifs in tandem and a DNA-binding (Mu) domain. Complexes prepared by combining plasmid DNA with TNM (DP) transfect MCF-7, COS, CHO and HepG2 cells. Ternary complexes prepared with DNA, protein and cationic lipid (DPL) resulted in ~5-7 fold enhancement in reporter gene expression over the DP alone. Treatment of cells with chloroquine during transfection, with DP complexes, resulted in remarkable increases in reporter gene expression suggesting the involvement of endosomal compartments in the uptake process. Interestingly, DPL prepared with Lipofectin or 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) exhibited enhanced transfection in the presence of serum in MCF-7 and HepG2 cells. Microinjection of DP complexes, with and without NLS sequence, into the cytoplasm and nucleus of smooth muscle cells (SMC) indicated that the presence of NLS sequence in protein carrier significantly enhanced transgene expression. Together the data suggest that modular design of proteins is a promising method to develop gene delivery carriers and also the role of NLS epitopes in mediating nuclear transfer of DNA complexes into various cell types.
Xavier
Jennifer
J
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.
Singh
Shashi
S
Dean
David A
DA
Rao
N Madhusudhana
NM
Gopal
Vijaya
V
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D002738:Chloroquine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D044967:Serum / Q000737:chemistry; D013499:Surface Properties; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D004268:DNA-Binding Proteins; D005229:Fatty Acids, Monounsaturated; D019913:Nuclear Localization Signals; D009696:Nucleic Acids; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine; D002738:Chloroquine; D001616:beta-Galactosidase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane